The general objective of the proposed research is to combine a new computational approach with complementary NMR methods for characterizing binding of small ligands to proteins. This will address a wide need for defining interactions of proteins with natural ligands, such as peptides, non-peptidic biomolecules, or with non-natural ligands, including drug leads or other chemicals. While this Component 2 is on interactions of small ligands with proteins, this approach can readily be applied to protein-protein or protein-nucleic acid interactions. This connects this research to Components 1 and 4. Thus, both aspects address the problem of characterizing weak but specific iinteractions of proteins with small molecules and other macromolecules in situations where experimental constraints are sparse. We proposeto develop an approach that will build on the recently developed TreeDock algorithm and will combine it with information from NMR spectroscopy on small-molecule inhibitors of protein-protein interactions obtained from high-throughput library screening. To define structures of weak protein-inhibitor complexes, TreeDock can search the docking space very fast by eliminating up front all configurations in which the components to be docked have no contact or penetrate each other. This is achieved with kd search trees. The algorithm uses complete enumeration of the search space at high resolution and can handle ligand mobility by querying a database of docked rigid subgroups. TreeDock can be ideally interfaced with sparse data from NMR experiments. We propose NMR methods that can provide few specific protein-ligand contacts even under difficult experimental conditions, such as intermediate exchange or poor solubility. The combination of the computational and experimental NMR methods will also be capable of dealing with structural changes of receptor proteins upon ligand binding. The research will be pursued four Specific Aims: 1: Development of a flexible docking algorithm (TreeDock II) that is capable of screening compound libraries efficiently, docking peptides and DNA/RNA to proteins. 2: Development of a compiler (parser, classifier) of chemical compound libraries. 3. Experimental methods for measuring NOEs in the intermediate exchange regime and defining anchors for TreeDock - Application to inhibitors of Bcl-2 type proteins. 4. Define complexes of small-molecule inhibitors with translation initiation factors 4E and 4A.